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enriched by a pressure against the counter-selection marker. This step, however,
is error-prone. First of all, there is no counter-selection system known that works
with 100% efficiency. This constitutive leakiness of any counter-selection pro-
vides a background that can be critical because the efficiency of the ET/red
recombination is low. Second, any counter-selection marker can mutate and the
mutated alleles will also appear as a background in large-scale cultures of bacte-
ria proficient for recombination. Third, the selection is applied for the loss of the
marker; therefore any unwanted rearrangement induced by repeated sequences
in the target genome leading to the loss of the marker will produce selectable
recombinants. The first two problems are associated with the counter-selection
system used. There are two procedures that seem to be efficient enough for
recombineering herpesvirus BACs, namely galK and the I-SceI meganuclease-
based counter-selection (Warming et al. 2005; Tischer et al. 2006). The risk of
genome rearrangements is controlled at best by the fine-tuning of recombinase
expression. However, the instability of the BACs is also influenced by the repeat
regions within the specific target genomes (Adler et al. 2000; Warming et al.
2005). Unfortunately, herpesvirus BACs abound in repeats of any kind. Not
surprisingly, the approaches of traceless recombineering have been tailored to
these genomes only long after the first reports on linear fragment mutagenesis of
MCMV BAC (Tischer et al. 2006).
Transposon Mutagenesis for Reverse and Forward Genetics
Transposons (Tns) are mobile genetic elements that insert themselves into a DNA
molecule (Craig 1997). After transfer of a Tn-donor plasmid into E. coli , the Tn can
jump into the viral BAC (Fig. 4). The temperature-dependent suicide Tn donor
plasmid is eliminated at the restrictive temperature. Some Tns preferentially insert
into the negatively coiled plasmids (e.g., Tn1721) and allow direct isolation of
mutated BACs (Brune et al. 1999). Others, like Tn5 or Tn10, are less selective and
mutated BACs need to be enriched by a retransformation round (Smith and Enquist
1999). The Tn insertion is determined by sequencing from primer sites within the
Tn (Brune et al. 1999) (Fig. 4b). Large libraries of mutant BAC genomes can
be established and screened for mutants of specific genes or gene families (Fig. 4c).
Even a comprehensive library of transposon mutants classified all known genes of
HCMV and guinea pig CMV with regard to their influence on the virus growth in
vitro (Yu et al. 2003; McGregor et al. 2004).
A support for genetics applications based on large libraries of randomly gener-
ated Tn insertion mutants is the usage of invasive bacteria as vehicles of virus
reconstitution. Certain Salmonella strains and E. coli strains expressing the bacte-
rial gene invasin and listeriolysin can invade mammalian cells and release plasmids.
Experimental transfer of a engineered plasmid-encoded transcription units by inva-
sive bacteria to mammalian cells has been shown both in vitro and in vivo (Darji
et al. 1997; Grillot-Courvalin et al. 1998). Accordingly, the MCMV-BAC was
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